Investigation of cytotoxic activity in four stachys species from iran.

The aerial parts of Stachys laxa Boiss. and Buhse. from Siah-bishe in Mazandaran province, Stachys trinervis Aitch. and Hemsl. from Karaj in Alborz province, Stachys subaphylla Rech. F. and Stachys turcomanica Trautv. from Golestan province have been collected in May 2008. Total extracts were obtained through MeOH/H2O (80/20) and then partitioned between CHCl3, EtOAc and MeOH. These fractions and total extracts have been investigated for in-vitro cytotoxic activity against the colon carcinoma (HT-29), colorectal adenocarcinoma (Caco-2), breast ductal carcinoma (T47D) and Swiss mouse embryo fibroblast (NIH 3T3) cell lines using MTT assay (3-(4,5-di methyl thiazol-2-yl)-2,5-di phenyltetrazolium bromide). At each cell line, doses of 3.125, 6.25, 12.5, 25, 100, 200, 400 and 800 µg/mL in 1% (v/v) DMSO of all samples were tested. Ethyl acetate and chloroform fractions of Stachys laxa against proliferation of T47D and HT-29 cell lines and chloroform fraction of Stachys subaphylla and Stachys subaphylla ethyl acetate fraction toward T47D cell line exhibited highest cytotoxic activity (IC50 < 50 µg/mL). Ethyl acetate and chloroform fractions of Stachys turcomanica against HT-29 cell line, except methanol fraction of Stachys subaphylla, the other extrcts on T47D cell line, represented moderate cytotoxic activity (IC50 < 70 µg/mL). All fractions of S. trinervis demonstrated no effective cytotoxic activity. IC50 values confirmed that the growth and proliferation of HT-29 and T47D cells were most affected by chloroform and ethyl acetate fractions of Stachys laxa and Stachys turcomanica due to their nonpolar compounds.


Introduction
The genus Stachys belongs to the plant family of Lamiaceae. The most species of this genus has been previously analyzed in numerous studies concerning their chemical composition, pharmacological properties and therapeutic uses. This family is well represented in the flora of Iran, at least with 200-300 species in the world (1) and 34 species in Iran (2). Phytochemical investigation of some Stachys species has demonstrated phenolic acids, tannins (3, 4), flavonoids (5) and phenyl ethanoid glycosides (6, 7). There are some reports about pharmacological activities of this genus including anticancer (8, were collected in May 2008. The plants have been identified and deposited at the Herbarium of Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.

Extraction
Freshly collected aerial parts of four species of Stachys were cleaned and shade dried. These parts were coarse powdered in a hand mill and stored at room temperature. Two hundred grams of powdered plants were extracted through perculation method with 80% aq. MeOH three times at room temperature. The extract was evaporated using rotary evaporator and consequently partitioned between CHCl 3 , EtOAc and MeOH. Each fraction evaporated with rotary evaporator and has been stored at refrigerator for the investigation of cytotoxic activity.

Plant material
The aerial parts of S. laxa Boiss. and Buhse., from Siah-bishe in Mazandaran province, S. trinervis Aitch. and Hemsl from Karaj in Alborz province, and S. subaphylla Rech. F. and S. turcomanica Trautv. from Golestan province (Anthos, Austria). The cell viability in MTT assay was calculated as the percentage of control value. Methotrexate was used as the positive control. Cytotoxicity was expressed as the concentration of extract inhibiting cell growth with 50% (IC 50 ± SD). All tests and analysis were run in triplicate.

Statistical analysis
IC 50 (the median growth inhibitory concentration) values were calculated from the IC 50 of dose-response curve in the sigma plot 11 software. Data representative of three independent experiments with similar results were presented as mean ± SD.

Result
The effects of these plant extracts on the proliferative response of the HT-29, Caco-2 and T47D cell lines have been analyzed by treating the cells with different concentrations of the extracts and significant decrease in cell lines proliferation were observed. IC 50 ± SD are reported in Table 1. The chloroform and ethyl acetate fractions of S. laxa Boiss. showed high cytotoxicity on T47D, HT-29 (IC 50 < 50 µg/mL). The cytotoxicity of ethyl acetate fraction of S. turcomanica Trautv. and chloroform fraction of S. subaphylla were better than the other fractions on T47D cell line (IC 50 < 50 µg/mL). Total extract and fractions of S. trinervis did not affect the cell lines.

Discussion
Among all the samples, nonpolar (chloroform and ethyl acetate fractions) fractions of S. laxa exhibited greatest cytotoxicities on T47D and HT-29 cell lines compared with polar fraction and total extract. According to the data, the